Due to its low cost, compatibility with automation and ease of scale-up, E. coli remains the most widely used host for high-throughput protein production (Hedren et al., Acta Cryst. 62 (2006) 1227-1231; Goulding and Perry, J. Structural Biol. 142 (2003) 133-143; Cabrita et al., BMC Biotechnology 6 (2006)). A major hurdle for heterologous protein production in E. coli is the formation of insoluble aggregates. This problem is commonly addressed through the use of fusion tags to enhance solubility (Waugh, TRENDS in Biotechnology 23 (2006) 316-320; Terpe, Appl. Microbiol Biotechnol 60 (2003) 523-533; Esposito and Chatterjee, Curr. Opin. Biotechnology, 17 (2006) 353-358). Comparative studies of the effectiveness of fusion tags have shown the maltose-binding protein (MBP) to be one of the best at solubilizing passenger proteins (Kapust and Waugh, Protein Science 8 (1999) 1668-1674; Hammarstrom et al., Potein Science 11 (2002) 313-321). The properties that make a fusion tag capable of enhancing solubility are not fully understood, although the acidity of the fusion tag is often correlated with this capability (Fox et al., FEBS Letters 537 (2003) 53-57; Su et al., J. Biotechnology 129 (2007) 373-382). Due to MBP's solubilizing capability and its affinity for amylose, which allow it to be used as an affinity handle, vectors containing MBP fusion tags have been developed for use in high-throughput cloning and expression (Donnelly et al., Protein Expr. Purif. 47 (2006) 446-454).
Although MBP is quite effective in solubilizing its passenger proteins during expression, a number of problems have been identified with its use. These problems can occur during purification and processing of the fusion. MBP fusions do not always bind to amylose resin and so a His tag is commonly added to facilitate affinity purification using a metal chelating resin (Pryor and Leiting, Protein Expr. Purif. 10 (1997) 309-319). Many proteins that are soluble when fused to MBP have been observed to precipitate when the MBP-fusion is cleaved (Donnelly et al., supra). Additionally, the incomplete removal of MBP from the passenger protein after cleavage of the fusion (Donnelly et al., supra) may interfere with downstream applications such as NMR or crystallization.
Thus, what is needed are improved fusion partners for solubilization and purification of proteins of interest.